PROJECT SUMMARY Spinocerebellar ataxia type 12 (SCA12) is a rare neurodegenerative disease caused by a CAG/CTG expansion in exon 7 of PPP2R2B, a gene encoding regulatory units of the protein phosphatase 2A. SCA12 is characterized by tremor, gait abnormalities, and psychiatric syndromes. Neuropathologically, the single SCA12 brain that has become available revealed prominent atrophy of the cerebral cortex and cerebellum, with a noted loss of Purkinje cells consistent with MRI findings in multiple pedigrees. PPP2R2B transcript is alternatively spliced with at least eight variants, each with a different N-terminal region. The repeat in exon 7 is itself included in at least three splice variants. In two variants, the repeat falls into ORFs predicted to encode the poly serine (polySer) tracts, giving rise to short polySer tracts from the normal allele and long polySer tracts from the expanded allele. Our preliminary data from cell systems suggests that even in the absence of AUG start codons, long polySer may be produced by repeat associated non-AUG (RAN) translation, and that proteins with a long polySer, but not a short polySer, have toxic properties. By CRISPR-assisted homologous recombination, we have successfully generated a humanized SCA12 knock-in (KI-10 or 80) mouse model, in which the mouse exon 2 was replaced by its human counterpart exon 7 with 10 or 80 CAG triplets. We hypothesize that the SCA12 KI-80 mouse model will recapitulate some features of the human SCA12 disease; and that poly amino acid tracts, especially long polySer, may be expressed in SCA12 KI-80, triggering toxicity hence SCA12 pathogenesis, and that the repeat expansion may alter PPP2R2B splicing and expression. In Aim 1, we will perform behavioral testing, neuropathology studies and neuroimaging to characterize the novel SCA12 KI-80 mouse model, along with its control SCA12 KI-10 model and WT. In Aim 2, we will determine the expression and potential toxicity of the predicted polySer containing proteins in SCA12 KI mice by correlating the polySer expression and aggregation with the progression of the mouse phenotype, behavior abnormalities and brain atrophy. We will examine the splicing and expression of PPP2R2B by RT-PCR and qPCR in KI-80, compared with KI-10 and WT. The proposed experiments provide some of the first steps in determining potential therapeutic targets for SCA12, and the appropriate models for testing rational therapeutics.